Recombinant human erythropoietin (r-HuEPO) is a relatively new treatment, useful in treating the anemia in renal failure patients in treatment with hemodialysis, however, it is necessary to supply iron and other nutrients to the patients because the requirements to iron and other nutrients increase when r-HuEPO is administered. Typically, in addition to r-HuEPO, a certain dose of intravenous iron is given, depending on the patient's iron status, to provide required iron supply. However, on the other hand, the toxicity of excess iron can pose life threatening risks to the patients. Careful evaluation of the iron status is of pivotal importance in end-stage renal disease patients before and during r-HuEPO therapy, which helps to determine appropriate treatment protocol.
In healthy individuals, iron concentrations in various tissues remain in a state of precise balance. Daily intake and loss of iron are small and body iron is reutilized. In patients with end-stage renal disease (ESRD), however, the supply of iron to the bone marrow may not be adequate to sustain normal erythropoiesis. Iron deficiency may also be caused by an increase in iron demand. Severe anemia associated with ESRD is mainly due to a deficiency in erythropoietin, a hormone produced by healthy kidneys. Replacement therapy with recombinant human erythropoietin, a stimulant of RBC formation, can correct this type of anemia in dialysis patients. However, r-HuEPO therapy increases the demand for iron. When iron stores cannot be mobilized quickly enough to be transported to the bone marrow where iron is needed for the production of new RBCs, a functional iron deficiency (FID) may result, despite an adequate iron supply. This functional iron deficiency can delay or diminish the response to r-HuEPO therapy. Iron supplementation is required to restore and maintain proper iron balance and to ensure optimal therapeutic response to r-HuEPO therapy.
Most recently, it has been reported that r-HuEPO is also a cytokine, which helps to cure the functional iron deficiency. Weiss et al. have shown that maintaining the serum ferritin of the ESRD patients higher than 150 ng/ml and only administering r-HuEPO without iron had no significant differences from the group of patients that received r-HuEPO/iron treatment during the follow up of three months. This has been attributed to the effect that r-HuEPO helps to liberate the iron that is blocked in the iron storage due to the inflammatory process (Weiss G, et al. Effect of iron treatment on circulating cytokine levels in ESRD patients receiving recombinant human erythropoietin. Kidney Int 2003; 64:572-8).
The most commonly used iron status parameters at present are transferrin saturation (TSAT) and serum ferritin (SF). However, both are indirect measures of iron status. Transferrin is a transport protein that contains two iron binding sites by which it transports iron from storage sites to erythroid precursors. TSAT (i.e., the percentage of total binding sites that are occupied by iron) is a measure of iron that is available for erythropoiesis. TSAT is calculated by dividing the serum iron by the total iron binding capacity (TIBC), a measurement of circulating transferrin, and multiplying by 100. Ferritin is a storage protein that is contained primarily within the reticuloendothelial system (RES), with some amounts released in the serum. Under conditions of iron excess, ferritin production increases to offset the increase in plasma iron. The level of ferritin in the serum, therefore, reflects the amount of iron in storage.
In normal individuals, SF levels range from 22 to 220 ng/ml and TSAT levels range from 20% to 40% (Suominen, P, et al., Serum Transferrin Receptor and Transferrin Receptor-Ferritin Index Identify Healthy Subjects With Subclinical Iron Deficits, Blood, Vol. 92, No. 8, 1998: pp 2934-2939). In patients without renal impairment, SF levels <22 ng/ml and TSAT <16% are indicative of depleted iron stores and absolute iron deficiency. In patients with chronic kidney disease, absolute iron deficiency is characterized by SF levels <100 ng/ml and TSAT <20%. Functional iron deficiency may be more difficult to diagnose since iron status parameters may indicate adequate iron stores. There are different criteria in defining FID, one of them is published by the Kidney Disease Outcomes Quality Initiative-K/DOQI (Eknoyan G, et al. Continuous quality improvement: DOQI becomes K/DOQI and is updated. National Kidney Foundation's Dialysis Outcomes Quality Initiative. Am J Kidney Dis., 2001 January;37(1):179-194), as shown in the following table.
Definition of Functional Iron Deficiency (FID) and Absolute IronDeficiency (AID) by Kidney Disease Outcomes, Quality InitiativeK/DOQI (U.S.A)Ferritin μg/l<100100-800TSAT <20%AIDTSAT 20%-50%FID
Because patients on r-HuEPO therapy may have adequate iron stores (as reflected by SF ≧100 ng/ml) but still have functional iron deficiency, the use of alternative iron parameters, for example, serum transferrin receptors (sTfR), alone or in combination with serum ferritin has been suggested in these patients (Weiss, G. et al. Review Article, Medical progress: Anemia of Chronic Disease. N Engl J Med 2005; 352:1011-23).
The reliability of using the serum ferritin to assess the iron status has been criticized, because serum ferritin is also a reactant phase protein, it is often elevated in the course of disease. Transferrin saturation has also been criticized, because the unreliability of serum iron measurements. Furthermore, transferrin saturation is affected by certain diseases, such as liver insufficiency, malnutrition, proteinuria, exudative enteropathy and acute phase reaction. In the past few years, numerous articles have reported that only using biochemical parameters to assess iron status is not sufficient for managing the r-HuEPO therapy.
Recently, the use of reticulocyte and red blood cell parameters has been suggested for detection of iron deficiency and for assistance in managing the r-HuEPO therapy. These new parameters include reticulocyte hemoglobin content (CHr) and percentage hypochromic red blood cells. More recently, RBC-Y (the mean value of the forward light scatter histogram within the mature erythrocyte population) and RET-Y (the mean value of the forward light scatter histogram within the reticulocyte population), which are obtained in the reticulocyte measurement on the SYSMEX® XE-2100 automated hematology analyzer have also been suggested.
Transferrin receptors on the cell surface of RBC precursors bind iron-bound transferrin, allowing the transport of iron from the plasma into the cells. Under conditions of iron deficiency, there is an upregulation of these receptors to allow more efficient uptake of transferrin. The concentration of transferrin receptors on the cell surface correlates with transferrin uptake. In hemodialysis patients who are not treated with r-HuEPO therapy, sTfR levels are higher among those who are iron deficient than among those who are iron replete. However, in several studies, hemodialysis patients treated with r-HuEPO therapy had similar sTfR levels regardless of iron status. Therefore, sTfR may not be an accurate marker of iron status in hemodialysis patients.
Reticulocytes are immature red blood cells (RBCs) with a life span of only 1 to 2 days. When these are first released from the bone marrow, measurement of their hemoglobin content can provide the amount of iron immediately available for erythropoiesis. A less than normal hemoglobin content in these reticulocytes is an indication of inadequate iron supply relative to demand. The amount of hemoglobin in these reticulocytes also corresponds to the amount of hemoglobin in mature RBCs. CHr is defined by the formula (CHr=MCVr×CHCMr), wherein MCVr is the mean reticulocyte cell volume and CHCMr is the mean hemoglobin concentration of reticulocytes which is obtained by an optical cell-by-cell hemoglobin measurement on the Bayer ADVIA 120 hematology analyzer. CHr has been evaluated in several studies as a test for functional iron deficiency and has been found to be highly sensitive and specific. However, exact threshold-values have not been established. Threshold values vary depending on the laboratory and instrument used.
Epoetin is effective in stimulating production of red blood cells, but without an adequate iron supply to bind to heme, the red blood cells will be hypochromic, i.e., low in hemoglobin content. Thus, in states of iron deficiency, a significant percentage of red blood cells leaving the bone marrow will have a low hemoglobin content. By measuring the percentage of red blood cells with hemoglobin content <28 g/dl, iron deficiency can be detected. Hypochromic red cells percentages >10% have been correlated with iron deficiency. Hypochromic red cell percentage (referred to as % Hypo) is reported by Bayer ADVIA® 120 hematology analyzer based on the optical cell-by-cell hemoglobin measurement.
Additionally, the red-cell distribution width (RDW) has been used in combination with other parameters for the classification of anemias. It reflects the variation in the size of the red cells and can be used to detect subtle degrees of anisocytosis. RDW is computed directly form the RBC histogram. Two different calculated values have been provided on hematology analyzers. The RDW-CV is measured as a ratio of the width of the distribution curve at one standard deviation divided by the MCV. The RDW-SD is a direct measurement of the distribution width at the 20% frequency level. Normally, the size distribution curve for red blood cells is quite symmetrical, with an RDW-CV value of 10±1.5% and an RDW-SD of 42±5 (fl). A high RDW, which means a greater variation in cell size, is caused by either the appearance of macrocytic or microcytic cells. An elevated red-cell distribution width appears to be the earliest hematological manifestation of iron deficiency.
As can be appreciated from the above, determining iron status, more particularly FID, is important for determining appropriate a treatment protocol. However, it is even more important and desirable, from a practical standpoint, if the doctors can effectively and reliably predict the patient's responsiveness to the r-HuEPO/intravenous iron treatment based on those available clinical chemistry and hematology parameters, as this can avoid unnecessary and expensive r-HuEPO therapy, and reduce the life threatening risks associated with inappropriate r-HuEPO/intravenous iron treatment given to the non-responders.